Flow cytometry has made it possible to analyze cells based on a variety of chemical and physical characteristics such as size, granulation of the cytoplasm and presentation of specific antigens. Flow cytometers, such as the FACScan.TM. instrument sold by Becton Dickinson Immunocytometry Systems (San Jose, Calif.) analyze cells on the basis of fluorescent and light scattering properties. To perform this analysis, cells are introduced into the center of a focused liquid stream which causes them to pass, one at a time, through the beam of a focused high power laser. Each cell is individually characterized by its light scatter signals and the intensity and color of fluorescence emitted in response to illumination by the laser.
In one type of cell sorting flow cytometer, after detecting the desired characteristic within an optic electronic system, the stream containing the individual cells is electrically charged shortly before being broken into droplets containing individual cells. An electrostatic field then diverts the flow of cell containing droplets into two or more streams depending on the polarity and charge of the droplets, and the divided streams are collected in separate containers. Particle sorting is described by M. J. Fulwyler in Science 150:910-911 (1965) and in IEEE Trans. on Nuclear Science, NS-21, pg. 714-720 (1973). Particle sorters which rely on electrostatic separation of particles are also described in U.S. Pat. Nos. 3,380,584; 3,710,933; 3,826,364; 4,148,718; and 4,230,558.
In another type of cell sorting flow cytometer, the particle stream is not broken into droplets for collection. Instead, the portion of the stream containing the cell of interest is collected after its detection by the optic system. This may be accomplished by deflection of the particle stream as in the fluidic switching flow sorter described by Duhnen, et. al. (Histochemistry (1983) 77:117-121) and in U.S. Pat. No. 4,175,662. Fluidic switching sorters, such as the PAS III instrument available from Partec (Muster, Germany), have a closed fluidic system and use gas controlled by piezoelectric valves to divert the fluid stream to sort cells. The closed fluidics also make this type of sorter preferable for use with infectious materials or samples which must be kept sterile.
Alternatively, the portion of the particle stream containing the desired particle may be collected by moving the catcher tube to align it with the stream at the appropriate time for collecting the particle. The catcher tube sorter does not rely on deflection of particles for sorting and therefore has the advantages of avoiding pressure pulses, minimizing damage to cells and reducing or eliminating biologically hazardous aerosols. This type of sorter is disclosed in U.S. Pat. No. 5,030,002. An example is the FACSort.TM. instrument (Becton Dickinson Immunocytometry Systems, San Jose, Calif.). This sorter is configured so that the catcher tube sits continuously in the edge of the fluid stream but is not aligned with the portion of the stream which contains cells until it is moved into the center of the stream to collect the desired cell. This sorter type therefore collects a relatively larger volume of fluid than does either the droplet sorter or the fluidic switching sorter, and the more rare the collected cells the more dilute the resulting cell suspension.
Cell recovery is therefore a particular problem when cells are sorted using catcher tube sorters such as the FACSort.TM., as these types of particle sorters tend to produce the sorted cells in a larger volume of fluid than either fluidic switching or droplet sorters. Recovery by centrifugation is inefficient when the cell suspension is dilute, and a substantial loss of cells often results. When very few cells are present in a large volume, recovery of any cells by centrifugation may be impossible. The present invention is therefore particularly useful for recovery of cells isolated in dilute solution by flow cytometry using closed sorting systems such as the FACSort.TM., but is also useful for recovering cells from dilute suspensions produced by other procedures.
Another useful application of the invention is in centrifugal cytology. In this procedure cells are sedimented out of liquid suspension onto slides for cytologic analysis. A centrifuge container, generally comprising a funnel, is used to hold the cell suspension during centrifugation and to direct the sedimented cells onto a discrete area of the slide. The Cytospin.TM. (Shandon) is an example of such a device. Loss of rare cells in dilute suspensions during centrifugation is also a problem in centrifugal cytology, presumably due to sticking to the walls of the funnel. Recovery of cells on slides using such procedures can be significantly improved using the methods of the invention.
Siliconizing the interior surfaces of centrifuge container is the art-recognized method for improving recovery of cells from liquid suspensions by centrifugation. Siliconization has several drawbacks, however, including the requirement for toxic chemicals such as dichlorodimethyl silane and chloroform to prepare the silicon coating. The procedure is also time consuming and cannot be used to coat polystyrene, a widely used centrifuge container material. The albumin coating methods of the invention, in contrast, are less toxic, easy to apply to a variety of materials including polystyrene, relatively inexpensive and are more effective for improving cell recovery than are silicon coatings.
Amphipathic molecules are those which contain both hydrophobic (nonpolar) regions and hydrophilic (polar regions. Proteins, fatty acids and surfactants are examples of amphipathic molecules. Of these, albumin is probably the best known in the art as a coating for a variety of surfaces and materials which come into contact with cells. R. A. Meck, et. al. (1980. Cytometry 1:84-86) describe albumin as a coating for microscope slides used to collect flow sorted cells. These authors report that fewer than half of the cells were retained on the albumin-coated slides after cytoloqical staining. R. Majuri, et. al. (1987. Eur. J. Haematol. 38:21 25) report that K562 cells do not attach to acrylic microbeads coated with albumin. Similarly, I. F. Charo, et. al. (1987. J. Biol. Chem. 262:9935-9938) teach that HUVE cells failed to adhere to albumin-coated glass slides. S. I. Rennard, et. al. (1983. Clin. Exp. Immunol. 54:239-247) disclose that no attachment of CHO cells occurred on albumin coated plastic petri dishes. F. Grinnell (1980. J. Cell Biol. 86:104-112) reported that latex beads coated with bovine albumin did not support binding of baby hamster kidney cells. V. P. Patel, et. al. (1985. PNAS 82:440 444) disclose that reticulocytes do not attach to albumin-coated plastic petri dishes. In contrast, D. Holderbaum, et. al. (1986. J. Cell. Physiol. 126:216-224) report attachment and growth of rabbit arterial smooth muscle cells to plastic culture dishes coated with bovine serum albumin. Therefore, although albumin is known in the art as a coating for a variety of surfaces and materials which come into contact with cells, such coatings were not previously known as a means to improve recovery of cells from liquid suspensions by centrifugation.
Block polymer surfactants, such as the Pluronic.sup..RTM. and Tetronic.sup..RTM. polyols (BASF Wyandotte Corp., Parsippany, NJ) are amphipathic molecules which comprise ethylene oxide and propylene oxide groups added to a base molecule (propylene glycol for Pluronic.sup..RTM. and ethylenediamine for Tetronic.sup..RTM.). The hydrophilic regions containing polyethylene oxide and the hydrophobic regions containing polypropylene oxide provide the surfactant properties. These surfactants and others like them are known in the art for foam control, emulsification, wetting, etc. They were not previously known as agents for improving recovery of cells from liquid suspension by centrifugation. Further, the utility of amphipathic molecules in general for this purpose has not previously been recognized.